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Folding and Glycosylation in the ER
Folding and Glycosylation in the ER The folding of proteins occurs in the lumen of the ER, where a variety of enzymes and chaperone proteins act to ensure correct folding of the newly synthesised proteins. Correct folding of newly synthesised proteins is necessary for the cell to recognise where the protein is destined for, and to allow for correct transportation of that protein. Protein folding also prevents aggregation of unfolded proteins, resulting in the accumulation of toxic material. The ER carries out four main post-translational modifications: *Removal of the signal sequence upon entry *Glycosylation *Disulphide bond formation *GPI addition GPI Addition In GPI addition, proteins destined for the the plasma membrane are covalently attached to a glycosylphosphatidyl-inositol (GPI) anchor at the c-terminus, which holds them in place. Preassembly of the GPI precursor occurs in the ER membrane, before the GPI molecule is added to the newly synthesised protein in the ER lumen. This linkage occurs while simultaneously, the transmembrane section of the membrane protein is cleaved off, so that the GPI anchor can replace it. (Cooper 2000) The GPI anchors newly synthesised proteins onto the luminal wall of the ER, and does so as soon as cleavage of the transmembrane section occurs. The membrane proteins are then released from the ER where they then travel to the cell surface in their soluble form and the GPI either anchors them in the plasma membrane or inserts them into lipid rafts in which they are separated from other membrane proteins (Alberts et al 2008, p.742-3). Glycosylation Glycosylation is the commonest modification performed by the ER. Almost half of the proteins in the ER, including the soluble and membrane bound proteins that are produced are glycosylated (Albert’s et al 2008, p.736) The purpose of glycosylation is to increase the stability of unfolded proteins, which prevents the degradation of proteins before they have had sufficient time to undergo folding. The glycosylation process begins with the addition of an oligosacharride chain to the unfolded proteins that consists of a mannose core (nine mannose molecules) with two N-acetylglucosamine residues that link it to phosphate dolichol and three glucose molecules on the other side of mannose core (Karp 2010, p.722) The oligosacharride chain is bound to the unfolded protein by the enzyme oligosaccharylstransferase, which creates an N-linked bond between the amine end of an asparagine residue and the N-acetylglucosamine molecule. Oligosacharride chains are bound to any asparagine amino acids present in the tripeptide sequences, Asn-X-Ser or Asn-X-thr, throughout the protein chain. These sequences are referred to as the glycosylation sequon. The oligosaccharide component of the molecule formed then undergoes a process known as glucose trimming, which removes two glucose and one mannose molecules from the chain. (Albert’s et al 2008, p.738). Protein folding and the CNX cycle Once the protein is modified, it is ready to undergo folding and enters a cyclical folding process, the main components of which are two chaperone proteins Calreticulin (CRT) and calnex (CNX). The first step in the cycle is the removal of three glucose molecules by the Glycosidase I and II enzymes. A third enzyme, UDP-glucose glycoprotein glycosyl transferase (UGGT) then adds one glucose molecule to the oligosacarride component of the unfolded protein. Addition of a glucose molecule enables the protein to interact with the two chaperone proteins CRT and CNX. The function of these chaperone proteins is to bind to the unfolded protein, retaining the protein in the ER, removing the protein’s ability aggregate with other unfolded proteins in the lumen; preventing any toxic material being formed, and to promote oxidative folding of the protein. (Anellia et all 2008) At regular intervals glucosidase II will remove the terminal glucose added by UGGT, causing the protein to dissociate from the CNX or CRT chaperone proteins. If the protein has still failed to fold correctly, UGGT adds another glucose molecule onto the unfolded protein to replace the glucose molecule removed by glucosidase II. This enables the unfolded protein to re-enter the CNX/CRT chaperones and the cycle is repeated. PF1.png|Three glucose molecules are removed by Glucosidase I and II enzymes before one glucose molecule is added to the protein by UDP. PF2.png|The glucose molecule added by UDP allows the unfolded protein to associate with the CRT chaperone. PF3.png|Glucosidase II then removes another glucose which causes the unfolded protein to dissociate from the CRT complex. PF4.png|If the protein has folded it is excreted from the ER. If the protein has not yet folded, UDP adds another glucose molecule allowing it to re-enter the folding cycle. Once the protein has folded correctly the protein dissociates from the CNX/CRT chaperones, ER mannosidase I interacts with the protein, removing a mannose molecle before it is recognised by highly specific lectin complexes, which assist in the exportation of the folded protein from the cell. However, Some proteins will never fold correctly, and if left unattended, would be stuck in the CNX/CRT cycle forever, needlessly clogging up the pathway and wasting the processing time of the cell. To prevent this, if a protein has been in the cycle for long periods of time, it will interact with ER Man I and II which removes a mannose molecule from the B branch of the oligosacharride component. The removal of mannose inhibits glucose re-addition, which in turn stops UGGT from interacting with the protein enabling the protein to re-enter the CNX/CRT cycle. Mannose cleavage is used to distinguish between young and old unfolded proteins, the younger ones should be given time to fold correctly, and therefore re-enter the cycle, while the older proteins; if still unfolded, should be targeted for degradation.